Throughout spermatogenesis, developing germ cells must remain attached to the seminiferous epithelium via testis-specific adherens junctions (AJ) between Sertoli and germ cells, such as ectoplasmic specializations and tubulobulbar complexes. The long-term objective of the PL's laboratory is to prepare male contraceptives that would perturb the functionality of these AJs thereby inducing the loss of cell adhesiveness, which in turn causes germ cells to deplete prematurely from the epithelium making them incapable of fertilizing the egg. Studies from this laboratory in the past decade have permitted the synthesis and identification of two potential male contraceptives: l-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) and 1 -(2,4,- dichlorobenzyl)-indazole-3-acrylic acid (AF-2785), having the effects of disrupting the adhesion of spermatids and spermatocytes onto the epithelium. Both AF-2364 and AF-2785 are effective to induce reversible male fertility in rats. They are neither nephrotoxic, hepatotoxic, nor affecting the hypothalamus-pituitary-testicular axis. Furthermore, recently completed acute toxicity studies and standard mutagenicity studies, which were performed by licensed toxicologists according to the FDA guidelines, have shown that both compounds are safe for further development. However, the mechanism(s) of action of these compounds at the cellular and molecular level is not known. We hereby propose two hypotheses to be tested by performing two series of mechanistic studies. First, AF-2364 exerts its effects exclusively on testis-specific AJs without affecting the blood-testis barrier (BTB) in the testis and AJs in other epithelia (Specific Aim 1). Second, AF-2364 and AF-2785 exert their effects by compromising the functionality of AJ-proteins (Specific Aim 2). Aim 1 will largely be in vivo studies investigating the effects of AF-2364 on the kinetics of spermatogenesis and AJ functionality using the techniques of histology, immunohistochemistry, micropuncture, and assays to assess the status and/or function of tight junctions (TJs) and AJs. Aim 2 will largely be in vitro studies to examine if these compounds exert their effects via the RhoB GTPase pathway, integrins, and other AJ-signaling molecules. These studies not only will unfold the site of action of either compound in the testis at the cellular and molecular levels; they will provide a framework to understand the biology and regulation of AJ dynamics in the testis. As such, additional targets, such as a specific signal transduction pathway pertinent to maintain the AJ functionality in the testis, will be identified to perturb germ cell attachment onto the epithelium. We also seek support to expand ongoing toxicity studies by performing 2 longterm toxicity studies in rats & dogs in anticipation of Phase I clinical Trials.